Exposure of bacteria to diverse growth-limiting stresses induces the synthesis of a large set of proteins that protect the cell against future, potentially lethal stresses. This general stress response brings about a special physiological state which enhances survival in the natural environment, in foods, and in some pathogenic interactions. Among Bacillus subtilis and related Gram positive pathogens, this response is governed by the ?B transcription factor. Loss of ?B function causes increased sensitivity to multiple stresses, including acid, antibiotic, cold, heat, osmotic, and oxidative stress. Our long term objective is to understand this response using B. subtilis as a model, beginning with the sensors which detect the different stresses, extending through the signal transduction network which conveys this information to ?B, and ending with the physiological role of genes under ?B control. This proposal addresses the signaling network itself, which functions by the partner switching mechanism in which key protein interactions are controlled by serine or threonine phosphorylation. This mechanism appears to be ancient, very plastic, and widespread among eubacteria. Study of this mechanism in B. subtilis should therefore help understand principles governing a broad array of signaling pathways. Here it activates ?B in response to two classes of stresses: (i) energy stress, including starvation for carbon, phosphate, or oxygen; and (ii) environmental stress, including acid, ethanol, heat, or salt. These two classes are conveyed to ?B by separate signaling pathways, each terminating with a differentially regulated PP2C phosphatase and each converging on the direct regulators of ?B, the RsbV anti-anti-? and the RsbW anti-?, which form one partner switching module. The energy branch consists of the RsbP phosphatase (with a PAS domain important for signaling) and RsbQ, a predicted hydrolase that is also required. The environmental branch has eight regulators, some redundant, and all join to activate the RsbU phosphatase via a second, atypical partner switching module. Six of these regulators co-purify from cell extracts in a large (>700 kDa) complex, and three form a simplified core amenable to analysis in vitro and in vivo. Our specific aims use a combined genetic, biochemical and structural approach to address three questions: (1) What is the mechanism of energy signaling; (2) What is the mechanism of environmental signaling; and (3) What is the role of positive and negative feedback in the ?B network?